Running Head: GENE BANKS AND WILDLIFE CONSERVATION

Gene Banks and their Roles in Wildlife Conservation Technology Environmental Stewardship Project Danielle Tentschert Miami University, Ohio

GENE BANKS AND WILDLIFE CONSERVATION Abstract Deforestation, pollution, habitat fragmentation, and poaching are just a few of the ways humans are negatively impacting the environment. As populations of species are

declining, so is their genetic diversity. Genetic bottlenecks are causing species to be more at risk for dying from diseases they do not have resistance to. Animals in artificial settings such as zoos and aquariums can help contribute to conservation and education by being a part of captive breeding programs through gene banks. Zoos that freeze genetic material can help save species from extinction and promote genetic diversity in both captive and wild populations. Material can be frozen for many years and then be thawed and used when needed to add more genetic variation, or simply produce more individuals to help keep a species surviving. This technology has also been used for storing seeds of various plant species to help replant more native species in areas taken over by invasive populations, reseed areas after fires, and prevent plant extinctions. This paper highlights the positive effects of zoo gene banking to show the important conservation work that is being done in such an environment. Keywords: Gene banking, DNA, conservation, frozen zoo, assisted breeding

GENE BANKS AND WILDLIFE CONSERVATION Introduction National parks, preserves, and wildlife reserves all function to save land in its natural state. They act as safe-havens for animals and plants which remain untouched,

surrounded by a world of environmental destruction and pollution. There are around eight thousand protected areas like these all over the world, yet approximately 96% of biodiversity remains unprotected (Wildt, 2000). It is estimated that there are currently somewhere between 10-100 million species that exist on Earth today (Holt & Pickard, 1999) with somewhere between 15,000 50,000 species are going extinct annually (Wildt, 2000). This can only be a rough estimate as many disappearing species have little known about them, and many extinctions are of species we have not even discovered yet (Holt & Pickard, 1999). Of the entirety of the known worlds species, less than 10% have had their conservation status determined. Of that 10%, the International Union for Conservation of Nature and Natural Resources (IUCN) has labeled 16000 species as threatened. Up to half of these 16000 species are amphibians, 23% are mammals, and 12% are birds (Clarke, 2009). Extinction of a species affects that species, the species above and below it in the food chain, and results in the loss of genetic information that evolution has taken millions of years to develop (Clarke, 2009). Extinction is a natural process, occurring slowly over many years. Human intervention including farming, over fishing, mining, water diversion and storage, industrialization, urbanization, etc. is causing fragmentation of habitats and environmental pollution (Holt & Pickard, 1999). Now extinction is occurring at an extremely high rate, driven by these unnatural causes. By building and fragmenting land, there is no longer enough habitat space to maintain biodiversity or genetic heterozygosity

GENE BANKS AND WILDLIFE CONSERVATION by finding mates from various packs with a different genetic makeup. Without this type of diversity, species cannot maintain healthy populations (Wildt, 2000). To help combat extinction and loss of genetic diversity, conservationists have taken to freezing genetic material to help save species and further scientific research and knowledge. Gene banks are generally assumed to only store semen and embryos (Boettcher, Stellar, Pizzi & Gandini, 2005), but they are much more than that. Anything

from tissues, somatic cells, gametes, eggs, sperm, and embryos can be stored and act as a back-up for when natural processes are not enough to keep a species thriving (Clarke, 2009). Zoos and aquariums are at the forefront of conservation because they act as preservations for some of the most threatened species in the world. The last remaining individuals of species seem to live and die in zoos, making zoos the first to know about a new extinction (Clarke, 2009). Action can be taken through genetic banks, which can help with breeding, saving species, promoting genetic diversity, and reintroducing species. The purpose of this paper is to highlight the important uses and processes of storing genetic material and zoo captive breeding programs. Discussion Genetic Losses By reducing species access to available mates, successful reproduction is hindered (Holt & Pickard, 1999). This reduced access to other individuals within a species and lack of natural bridges to move to other habitat areas causes inbreeding to be more common, leading to a loss of genetic diversity. Genetically inherited diseases, birth defects, and a lowered immune system are much more likely to occur due to inbreeding,

GENE BANKS AND WILDLIFE CONSERVATION which in turn reduces the fitness within the populations (Holt & Pickard, 1999). Loss of

genetic diversity also leads to susceptibility for a species to be wiped out by disease as all individuals would be at risk: none would have immunity or biological advantage over any other individuals in the species. Genetic diversity allows a species to survive and adapt within a changing environment. It provides variations in traits allowing adaptations in a population. More traits mean natural selection can help select the fittest individuals to survive; without it, an entire population could be wiped out by not having the necessary adaptations to cope with a specific change. Contaminants from businesses and manufacturing plants adversely affect animal reproduction. Sperm production has become less efficient, (Holt & Pickard, 1999) so even when animals try to mate and reproduce, the success rate of pregnancy is also declining. The loss of genetic variation causes genetic drift, demographic bottlenecks, founder effects, and inbreeding (Wildt, 2000). It is important to save the genetic material of threatened animal species before they become extinct (Clarke, 2009). History of gene banking Gene banking, sometimes known as cryopreservation, is the process of taking genetic material from animals, freezing, and storing that material until it can be used for future needs. Such banks are primarily used to promote biodiversity. Gene banking is not a new idea but the technology is recent and improving. It was suggested by the US National Academy of Sciences, back in 1978, that sperm and embryo freezing for domestic species should be applied to preservation of wild species (Wildt, 2000). It was not until 1995 that the Animal Gene Storage and Resource Centre of Australia created

GENE BANKS AND WILDLIFE CONSERVATION one of the first frozen storage facilities for threatened species. In 2000 it was proposed that an international database should be created to bank DNA and cells from threatened animals that the world could have access to. The UK Frozen Ark project was finally launched in 2005, a project for banking genes of as many different species as possible (Clarke, 2009). The race to sequence the entire human genome was only just recently completed and already there have been vast improvements in cost, efficiency, and time (Clarke, 2009). Humans have been using forms of gene banking in sperm and egg banks for their own needs of having children. Now conservation laboratories across the globe are working to use similar methods to help species populations that need help. Gene Banks Artificial insemination, embryo transfer, or in vitro fertilization are all ways in which genetic resource banks can be used. Banking genetic information essentially allows an individual to contribute to breeding programs even after they have died, thus lengthening their genetic lifespan (Holt & Pickard, 1999). To optimize success of such breeding programs, non-invasive strategies such as endocrine monitoring have been implemented into the gene banking process. By analyzing urine and feces, the

reproductive status of an animal can be better gauged. This can be useful for inseminating at the optimum time as well as diagnosing pregnancy (Holt & Pickard, 1999). As opposed to simply selecting specific males and females, genetic drift can be best managed by allowing all animals in the population to mate and contribute equally to the offspring in their population (Johnston & Lacy, 1995), therefore for a breeding program to be most successful, sperm should be collected from as many males as possible. After

GENE BANKS AND WILDLIFE CONSERVATION estrus synchronization and tracking of a females cycle, one can be anesthetized and laproscopically inseminated with the previously frozen sperm (Wildt, Monfort, Donoghue, Johnston, & Howard, 1992). Avoiding inbreeding can be tricky as sperm is used from all males in a group; as more successful pregnancies occur in offspring, halfsiblings will contribute to a decreased effective population size in future populations (Boettcher, Stellar, Pizzi & Gandini, 2005). Sperm donors must be tracked and parental tests can be used to help avoid any inbreeding of viable offspring.

Similar to the aforementioned Frozen Ark in the UK, San Diego Zoo Global has its own Frozen Zoo. This includes a collection of viable cell cultures, semen, embryos, oocytes and ova, blood, and DNA and tissue specimens, from over 10,000 individuals of 1,000 species (Ryder, 2014). The San Diego Zoo is using this museum of collected biological information to help with barcoding for species conservation, learn about chemicals that disrupt the endocrine system, and help with recovery of species. These in turn would help with the current amphibian extinction crisis and allow for paternity tests once a female becomes pregnant to avoid inbreeding with that offsprings future mating. Some of the laboratory methods used include cell culture, karyotyping, DNA isolation, and PCR amplification of nucleic acids. Such methods support further studies of the human genome, by providing an array of the genetic relatedness between many diverse mammal populations. Storage Cryopreservation is the process of freezing and preserving biological samples in a suspended state (Karlsson & Toner, 1996). Tissue, blood, or gametes need to be frozen and stored between -70 degrees Celsius to -196 degrees Celsius (the temperature of liquid

GENE BANKS AND WILDLIFE CONSERVATION nitrogen). This can cost around $600 USD for storage of each set of gametes or DNA from one species and, if stored at these temperatures, the genetic material could last for thousands of years (Clarke, 2009). The colder the temperature at which samples are stored, the longer they will last. The optimum temperature would be that of liquid nitrogen at -196 degrees Celsius, which gives a shelf life to the stored cells of 103 years (Karlsson & Toner, 1996). Challenges to gene banks As with any form of technology and especially with any conservation efforts, there are always challenges. Research can be expensive and difficult. Projects such as

The Frozen Zoo and The Frozen Ark need funding, staffing, and a storage space. Also, as bodily fluids and materials are being stored for future use on other animals, scientists must assess the risks of disease transmission from the samples (Holt & Pickard, 1999). Cell injury can occur due to the change in temperature and consequences from ice formation; the two major stressors of cryopreservation. The samples undergo large fluctuations between body temperature and below freezing, which can result in both cold shock and warm shock (Watson, 1995). Eggs are more likely to be damaged by the freezing process so most genetic resource banks focus on the storage of frozen semen (Clarke, 2009). Although embryo collection and freezing techniques are improving, cryopreservation is more difficult, more expensive, and more complex with embryos. Also, in terms of quantity, semen provides more chances of producing offspring in multiple eggs, but you can only harvest and impregnate one egg at a time from a female. To properly reconstruct a population from just frozen eggs, many more donor females would be needed, which would be hard to acquire in an already endangered species

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(Boettcher et al., 2005). Combining the use of semen and embryos allows more retention of the original genome of the breed and could allow for more successful pairings (Boettcher et al., 2005). Some countries do not have reliable sources of electricity to maintain freezer equipment. To remedy this potential problem, it has been found that DNA could be freeze dried and stored between room temperature to -20 degrees Celsius and last at least 30 years (Clarke, 2009). A big challenge is the lack of availability of diverse biological material to better understand wild populations reproductive patterns (Andrabi & Maxwell, 2007). There is variation between species, and the less we know about a species, the harder it is to successfully have breeding programs. There can also be some challenges to correctly tracking exact time of ovulation (Widlt et al., 1992) but many of these problems have been worked out through carefully tracking female cycles before artificial insemination. Seed Banks Storage of genetic material is not only done to save animal species. Many native plant species will decline or disappear within our lifetime. This will undoubtedly impact surrounding plant communities and animals that depend on them. Wildfires, climate change, and invasive species are negatively effecting native plant populations all over the globe. Frequency and intensity of fires in the United States are increasing and the few land patches that have not been built on by humans are being destroyed. Invasive grass species are dry and easily catch fire and there is a lack of native plant seeds to be able to properly restore the area (Ryder, 2014).

GENE BANKS AND WILDLIFE CONSERVATION All of the aforementioned environmental issues can be mitigated by seed banks. Seed banks store germplasm, living tissue from which new plants can be grown, for research and restoration. Projects such as the Seeds of Success Program collects seeds from native plants, (Ryder, 2014) which are then separated out and used for different

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purposes. Some are planted to cultivate more seeds, others used for restoration, and some are even banked in the U.S. National Seedbank to save for the future. The seed banking process consists of collecting the seeds, cleaning and drying them, identifying the type of seed, and then moving the seeds to either short or long-term storage. Periodic germination tests are conducted in order to determine viability of the seeds in storage. The San Diego Zoo has already collected and stored seeds of 170 species (Ryder, 2014). Conclusion In conclusion, zoo research is very important to conservation. Gene banking can help save species and promote genetic diversity but it alone is not the solution. Gene banks need to be combined with other conservation methods to be most successful. Freezing genetic material such as eggs, sperm, DNA, and tissues from animals can help us better understand populations and fight extinction. Storing plant seeds can not only help keep genetic diversity of plants and preserve a variety of species, but it can also help ensure the future of agriculture. The Frozen Zoo and the Frozen Ark are just two examples of this technology being implemented by zoos and conservationists around the world. In future research, it may be possible to look at how the genetic material change over time in the freezers and if it would be possible to create pregnancies from a frozen egg and frozen sperm within an incubator so that no stress at all would come to live

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animals. If that could become possible then we could bring animals back from extinction, but for now this technology can help us prevent any new extinctions.

GENE BANKS AND WILDLIFE CONSERVATION References

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Andrabi, S.M.H, & Maxwell, W.M.C. (2007) A review on reproductive biotechnologies for conservation of endangered mammalian species. Animal Reproduction Science, 99, 223-243. Boettcher, P.J., Stellar, A., Pizzi, F., & Gandini, G. (2005). The combined use of embryos and semen for cryogenic conservation of mammalian livestock genetic resources. Genetics Selection Evolution, 37, 657-675. Clarke, A. G. (2009). The frozen ark project: the role of zoos and aquariums in preserving the genetic material of threatened animals. International Zoo Yearbook, 43 (1), 222-230. Holt, W.V., & Pickard, A.R. (1999). Role of reproductive technologies and genetic resource banks in animal conservation. Reviews of Reproduction, 4, 143-150. Johnston, L.A., & Lacy, R.C. (1995). Genome resource banking for species conservation: selection of sperm donors. Cryobiology, 32, 68-77. Karlsson, J.O.M., & Toner, M. (1996). Long-term storage of tissues by cryopreservation: critical issues. Biomaterials, 17 (3), 243-256. Ryder, O. (2014). San Diego Zoo Institute for Conservation Research, Frozen Zoo. Retrieved from http://www.sandiegozooglobal.org/what_we_do_banking_genetic_resources Watson, P.F. (1995). Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproductive Fertilization Development, 7, 871-891.

GENE BANKS AND WILDLIFE CONSERVATION Wildt, D.E. (2000). Genome resource banking for wildlife research, management, and conservation. ILAR Journal, 41 (4), 228-234. Wildt, D.E., Monfort, S.L., Donoghue, A.M., Johnston, L.A., & Howard, JoGayle. (1992). Embryogenesis in conservation biology or, how to make an endangered species embryo. Theriogenology, 37 (1), 161-184.

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